| In sporophytically self-incompatible Brassica, three S-linked genes, the pollen ligand SCR, stigma receptor SLG and SRK, have been identified. In addition, four genes (ARC1, THL1, THL2 and MOD) that do not link to S locus have also been found, they are regarded as downstream protein factors of self-incompatibility (SI) response, however, their biological function is not clear. The self-incompatibility is involved in a signal transduction process resulted from the interaction between ligand and receptor. Up to now, some achievements for ligand and receptor have already been made, but the research progress on downstream protein factors is slower than that of upstream components, highlighting the downstream proteins in priority of research. Some downstream components have not been isolated, which are similar to the unknown function of those isolated ones.'Xiyuan4', a B. olereacea L. and '2001817', a B. napus L. were bred by Southwest Agricultural University. Here using them as materials, we studied and explored thoroughly the DNA and cDNA sequences of THL2 gene and MOD gene with bioinformatical methods. For the first time, based on sequence data, we analyzed the structure, heredity and variation of the DNA sequences encoding THL2 and MOD, and foresaw the molecular mechanism in self-incompatibility signal process. The main methods and results are as the followings:1. Cloning and sequence analysis of THL2 geneUsing the genomic DNA and stigma total RNA isolated from B.olereacea L. and B.napus L. as templates in PCR and RT-PCR reaction, the THL2 DNA and cDNA were amplified, recovered, cloned into vectors and sequenced. The results demonstrated that in B. napus L. THL2 DNA and cDNA were 888 bp and 524 bp in size respectively, similar to that of B. olereacea L. whose size were 879 bp and 516 bp respectively. Two introns were found for the first time in THL2 genomic DNA. Comparing THL2 DNA encoding region of B.olereacea L. with that of B. napus L., 4 different bases were found, and three of them led to the alteration of amino acids. Homologous analysis in nucleotide sequence using MegAlign program demonstrated that THL2 DNA from both B.napus L. and B.olereacea L. were 96% in identity. Homologous analysis in amino acid sequence showed that between THL2 in B.olereacea L. and TRX4 in Arabidopsis thaliana were 97% in identity, a little difference to that of 86% in identity between THL2 in B.napus L. and TRX4 inA.thaliana. Evolution analysis also indicated the similar conclusion that THL2 and TRX4 had a very close evolution relationship. Predicted tertiary structure showed that THL2 protein possessed a βαβ domain, and the SRK-binding site CPPC is located in tertiary structure surface, which provided the spacial foundation for THL2 binding SRK kinase.2. Cloning and sequence analysis of MOD geneUsing the genomic DNA and pistil total RNA isolated from B.olereacea L.and B.napus L. as templates in PCR and RT-PCR reaction, the MOD DNA and cDNA were amplified, recovered, cloned into vectors and sequenced. The results showed that MOD DNA and cDNA were 1378 bp and 853 bp in size respectively. For the first time, three introns were found in MOD genomic DNA by comparing DNA to cDNA , whose size were 315 bp, 123 bp and 87 bp respectively.Comparing the MOD cDNA of B.olereacea L. with that of B. napus L., 31different bases were found, and only one of them led to the alteration of amino acid residues based on the predicted amino acid sequences. Enzyme cutting analysis indicated that they had different enzyme cutting sites and cutting site number. The homologous analysis in amino acid sequence showed that between MOD in B.olereacea L. and PIP1b, PIP1.1, PIP1.3, PIP1.4, PIP1.5 in A thaliana were 99%, 97% , 94%, 94%, 91% in identity respectively, but both MOD in B.olereacea L. and TIP2.1 vacuole membrane protein in A.thaliana were only 37% in identity. Evolution analysis also resulted in the similar conclusion.Using the online Scanprosite program, some phosphorylation sites on MOD protein were found for the first t...
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